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US20050032081A1 - Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry - Google Patents

Biomolecular coupling methods using 1,3-dipolar cycloaddition chemistry Download PDF

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US20050032081A1
US20050032081A1 US10/735,081 US73508103A US2005032081A1 US 20050032081 A1 US20050032081 A1 US 20050032081A1 US 73508103 A US73508103 A US 73508103A US 2005032081 A1 US2005032081 A1 US 2005032081A1
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biomolecule
group
covalently
azido
molecule
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Jingyue Ju
Tae Seo
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Columbia University in the City of New York
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • Synthetic oligonucleotides are the most important molecular tools for genomic research and biotechnology (1). Modified oligonucleotides are widely used as primers for DNA sequencing (2) and polymerase chain reaction (3), antisense agents for therapeutic applications (4), molecular beacons for detecting genetic mutations (5), and probes for measuring gene expression in DNA microarrays and gene chips (6).
  • the modification of either the 3′- and 5′-termini or an internal position of the oligonucleotides with a primary alkyl amine group is a widely used method for introducing additional functional groups to DNA (7). Introduction of these functionalities to DNA can be achieved through the use of appropriate phosphoramidite reagents in solid phase synthesis. Once a unique functional group is incorporated into the DNA, the functional group can subsequently be conjugated to the desired molecule by a selective chemical reaction.
  • succinimidyl ester of a fluorescent dye is widely used to couple with a primary amine group introduced to an oligonucleotide (8).
  • the coupling reaction requires aqueous conditions that can hydrolyze the succinimidyl ester moiety.
  • phosphoramidite derivatives of fluorescent dyes were used to directly couple with the oligonucleotide in the solid phase synthesis (9).
  • the functional group is labile to the basic deprotection conditions used in solid phase DNA synthesis, the direct phosphoramidite approach cannot be used.
  • coupling chemistry with high stability and high yield to modify DNA and other biomolecules.
  • chemoselective modification of protein and cell surfaces by the Staudinger ligation has been developed (10), and the Diels Alder reaction was also explored for the selective immobilization of proteins (11).
  • Ideal coupling functional groups (one on the DNA and the other on the molecule to be coupled) should be stable under aqueous reaction conditions.
  • the coupling reaction should be highly chemoselective with a high yield, and the resulting linkage should be stable under biological conditions.
  • click chemistry as a set of powerful, highly reliable, and selective reactions for the rapid synthesis of useful new compounds and combinatorial libraries through heteroatom links (12).
  • One of the click chemistry reactions involves the coupling between azides and alkynyl/alkynes to form the triazole version of Huisgen's [2+3] cycloaddition family (13).
  • Mock et al. (14) discovered that cucurbituril could catalyze this 1,3-dipolar cycloaddition.
  • This coupling chemistry was also used to form oligotriazoles and rotaxanes by Steinke et al. (15). The addition results in regioisomeric five-membered heterocycles (16).
  • This invention provides a first method for covalently affixing a biomolecule to a second molecule comprising contacting a biomolecule having an azido group covalently and operably affixed thereto with a second molecule having an alkynyl group covalently and operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the azido and alkynyl groups, thereby covalently affixing the biomolecule to the second molecule.
  • This invention also provides a second method for covalently affixing a biomolecule to a second molecule comprising contacting a biomolecule having an alkynyl group covalently and operably affixed thereto with a second molecule having an azido group covalently and operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the alkynyl and azido groups, thereby covalently affixing the biomolecule to the second molecule.
  • This invention also provides a first method for covalently affixing a biomolecule to a solid surface comprising contacting a biomolecule having an azido group covalently and operably affixed thereto with a solid surface having an alkynyl group operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the azido and alkynyl groups, thereby covalently affixing the biomolecule to the solid surface.
  • This invention further provides a second method for covalently affixing a biomolecule to a solid surface comprising contacting a biomolecule having an alkynyl group covalently and operably affixed thereto with a solid surface having an azido group operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the alkynyl and azido groups, thereby covalently affixing the biomolecule to the solid surface.
  • This invention further provides a biomolecule having either an azido group or an alkynyl group covalently and operably affixed thereto.
  • This invention further provides a solid surface having an azido group or an alkynyl group operably affixed thereto.
  • This invention provides a biomolecule covalently affixed to a second molecule via one of the instant methods.
  • This invention further provides a biomolecule covalently affixed to a solid surface via one of the instant methods.
  • This invention further provides a biomolecule covalently affixed to a second molecule via a 1,2,3-triazole ring.
  • this invention further provides a biomolecule covalently affixed to a solid surface via a 1,2,3-triazole ring.
  • FIG. 1 Scheme for synthesizing an oligonucleotide labeled by an azido group at the 5′ end.
  • FIG. 2 MALDI-TOF mass spectrum of structure 2 of FIG. 1 .
  • FIG. 3 Scheme showing 1,3-dipolar cycloaddition between alkynyl-FAM and azido-labeled DNA.
  • FIG. 4 MALDI-TOF MS spectrum of structures 4 and 5 of FIG. 3 .
  • FIG. 5 Electropherogram of the DNA sequencing fragments generated with structures 4 and 5.
  • FIG. 6 Immobilization of a polypeptide on a solid surface.
  • FIG. 7 Immobilization of a polypeptide on a solid surface.
  • FIG. 8 Immobilization of a polysaccharide on a solid surface.
  • FIG. 9 Immobilization of protein on a solid surface.
  • FIG. 10 Immobilization of an oligonucleotide on a solid surface.
  • FIG. 11 Immobilization of DNA on a glass surface in the presence of Cu(I) Catalyst.
  • Antibody shall include, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, this term includes polyclonal and monoclonal antibodies, and fragments thereof. Furthermore, this term includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.
  • Biomolecule shall mean a molecule occurring in a living system or non-naturally occurring analogs thereof, including, for example, amino acids, peptides, oligopeptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, nucleic acids, DNA, RNA, lipids, enzymes, receptors and receptor ligand-binding portions thereof.
  • Carbohydrate shall mean an aldehyde or ketone derivative of a polyhydroxy alcohol that is synthesized by living cells, and includes monosaccharides, disaccharides, oligosaccharides, and polysaccharides synthesized from saccharide monomers.
  • Covalently affixing shall mean the joining of two moieties, via a covalent bond.
  • Lipid shall mean a hydrophobic organic molecule including, but not limited to, a steroid, a fat, a fatty acid, or a phospholipid.
  • Nucleic acid shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof.
  • the nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).
  • “Operably affixed” in reference to an azido group or an alkynyl group shall mean that the group is affixed to a molecule or surface in such a way as to permit the azido or alkynyl group to undergo a 1,3-dipolar cycloaddition with an alkynyl or azido group, respectively, on a different molecule or surface, as applicable.
  • R n in an embodiment where the biomolecule is a peptide, can be a side chain of n amino acids.
  • Each repeating unit is, for example, one of 20 amino acids or their analogues, and shall include e.g. Glycine, Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tyrosine, Tryptophan, Serine, Threonine, Cysteine, Methionine, Asparagine, Glutamine, Aspartate, Glutamate, Lysine, Arginine, Histidine. Lysine, Arginine, Serine, Cysteine, or Threonine is preferred as the carboxyl-terminal residue.
  • n can be, for example, 1-500.
  • the azido or alkynyl functional group is located at the terminal sugar ring.
  • R is a hydrogen for DNA and a hydroxyl group for RNA
  • N is, for example, 1-200.
  • B groups are heterocyclic ring systems called bases. The principal bases are adenine, guanine, cytosine, thymine, and uracil.
  • the biomolecule is a protein, for example, an enzyme, antigen, or antibody
  • the positions of the azido and the alkynyl functional groups are easily interchangeable.
  • X can be, for example, an aliphatic or aliphatic-substituted derivative, aryl or aryl-substituted group, electron-withdrawing functional group or electron-releasing group.
  • An aliphatic chain shall include, for example, a lower alkyl group, in particular C 1 -C 5 alkyl, which is unsubstituted or mono- or polysubstituted, e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, or n-pentyl.
  • An aryl or aryl-substituted group shall include, for example, a phenyl, or an o-, m-, p-substituted phenyl, e.g. p-methylphenyl, p-chlorophenyl, p-nitrophenyl group.
  • An electron-withdrawing functional group shall include, for example, an alkoxy substituted alkyl, e.g. diethoxymethyl, or halogenated carbon substituent, e.g. chloromethyl, trifluoromethyl, or an alkyl ester, e.g. methyl ester, ethyl ester, or a ketone derivative, e.g.
  • An electron-releasing group shall include, for example, an alkoxy group, e.g. methoxy, ethoxy, or an alkylamino group, e.g. diethylamino, phenylmethylamino.
  • This invention provides a first method for covalently affixing a biomolecule to a second molecule comprising contacting a biomolecule having an azido group covalently and operably affixed thereto with a second molecule having an alkynyl group covalently and operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the azido and alkynyl groups, thereby covalently affixing the biomolecule to the second molecule.
  • This invention also provides a second method for covalently affixing a biomolecule to a second molecule comprising contacting a biomolecule having an alkynyl group covalently and operably affixed thereto with a second molecule having an azido group covalently and operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the alkynyl and azido groups, thereby covalently affixing the biomolecule to the second molecule.
  • the biomolecule can be, for example, a nucleic acid, a protein, a peptide, a carbohydrate, or a lipid.
  • the biomolecule is DNA, an antibody, an enzyme, or a receptor or a ligand-binding portion thereof.
  • the biomolecule can be a nucleotide, an oligonucleotide, a polynucleotide, a lipid, a lipid derivative, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide.
  • the second molecule can be, for example, a biomolecule, a fluorescent label, a radiolabeled molecule, a dye, a chromophore, an affinity label, an antibody, biotin, streptavidin, a metabolite, a mass tag, or a dextran.
  • the biomolecule can be a nucleotide, an oligonucleotide, a polynucleotide, a lipid, a lipid derivative, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a monosaccharide, a disaccharide, an oligosaccharide, or a polysaccharide.
  • the biomolecule is immobilized.
  • the second molecule is immobilized.
  • neither the biomolecule nor the second molecule is immobilized.
  • Conditions permitting a 1,3-dipolar cycloaddition reaction to occur are known, and can comprise for example, the application of heat, contacting at room temperature, and contacting at 4° C.
  • the contacting is performed in the presence of an agent which catalyzes a 1,3-dipolar cycloaddition reaction.
  • the reaction is carried about within the temperature range 50° C. to 150° C., and more usually at between 70° C. to 100° C.
  • the molar ratio of cataylyst:alkynyl group:azido group is from 0:1:1 to 2:1:100, and preferably 1:1:0.5.
  • the reaction is carried out in the aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • the molar ratio between the alkynyl group and the azido group is from 1:1 to 1:100.
  • a catalyst such as a Cu(I) catalyst, the reaction may be performed at room temperature.
  • This invention also provides a first method for covalently affixing a biomolecule to a solid surface comprising contacting a biomolecule having an azido group covalently and operably affixed thereto with a solid surface having an alkynyl group operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the azido and alkynyl groups, thereby covalently affixing the biomolecule to the solid surface.
  • This invention further provides a second method for covalently affixing a biomolecule to a solid surface comprising contacting a biomolecule having an alkynyl group covalently and operably affixed thereto with a solid surface having an azido group operably affixed thereto under conditions permitting a 1,3-dipolar cycloaddition reaction to occur between the alkynyl and azido groups, thereby covalently affixing the biomolecule to the solid surface.
  • biomolecules and reaction conditions are the same as those set forth above in connection with the first and second methods for affixing a biomolecule to a second molecule.
  • the solid surface can be, for example, glass, silica, diamond, quartz, gold, silver, metal, polypropylene, or plastic.
  • the solid surface is silica.
  • the solid surface can be present, for example, on a bead, a chip, a wafer, a filter, a fiber, a porous media, or a column.
  • This invention further provides a biomolecule having either an azido group or an alkynyl group covalently and operably affixed thereto.
  • This biomolecule can be, for example, a nucleic acid, a protein, a peptide, a carbohydrate, or a lipid.
  • the biomolecule is DNA.
  • This invention further provides a solid surface having an azido group or an alkynyl group operably affixed thereto.
  • This solid surface can be, for example, glass, silica, diamond, quartz, gold, silver, metal, polypropylene, or plastic.
  • the solid surface can be, for example, present on a bead, a chip, a wafer, a filter, a fiber, a porous media, or a column.
  • the solid surface is a silica surface.
  • the silica surface is part of a chip.
  • This invention provides a biomolecule covalently affixed to a second molecule via one of the instant methods.
  • This invention further provides a biomolecule covalently affixed to a solid surface via one of the instant methods.
  • This invention further provides a DNA molecule covalently attached to a glass surface via one of the instant methods.
  • This invention further provides a biomolecule covalently affixed to a second molecule via a 1,2,3-triazole ring.
  • this invention further provides a biomolecule covalently affixed to a solid surface via a 1,2,3-triazole ring.
  • oligonucleotide labeled by an azido group at the 5′ end as shown in FIG. 1 .
  • 5-Azidovaleric acid was synthesized according to the literature (18) and activated as N-succinimidyl ester “1” (87%).
  • the oligonucleotide 5′-amino-GTT TTC CCA GTC ACG ACG-3′ was reacted with excess succinimidyl 5-azidovalerate “1” to produce the azido-labeled DNA “2” (see FIG. 1 ).
  • FIG. 2 shows the MALDI-TOF MS spectrum of the isolated product, with a single major peak at 5757 Da that matched very well with the calculated value of 5758 Da for the azido-DNA 2. This indicates that the starting material amino-DNA was quantitatively converted to the azido-DNA 2 (coupling yield ⁇ 96%).
  • the primer synthesized by the click chemistry can be used directly to produce DNA sequencing products with singe base resolution in a capillary electrophoresis DNA sequencer with laser induced fluorescence detection.
  • a reduced reaction time can be achieved by attaching an electron withdrawing functional group at the end of the triple bond (12).
  • FIG. 6 shows the immobilization of a polypeptide on a solid surface by 1,3-dipolar cycloaddition reaction.
  • the polypeptide is labeled with an azido group at the carboxyl-terminal residue, while the solid surface is modified by a heterobifunctional linker which produces a substituted alkynyl group at the end.
  • the polypeptide is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • FIG. 7 shows the scheme for the immobilization of a polypeptide on a solid surface by 1,3-dipolar cycloaddition reaction.
  • the polypeptide is labeled with a substituted alkynyl group at the carboxyl-terminal residue, while the solid surface is modified by a heterobifunctional linker which produces an azido group at the end.
  • the polypeptide is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • the 1,3-dipolar cycloadditon reaction is controlled either thermodynamically at high temperature, or catalytically at room temperature with cucurbituril (21). In the absence of the catalyst, the reaction is carried about within the temperature range 50° C. to 150° C., and more usually at between 70° C. to 100° C.
  • the reaction takes from 5 hours to 7 days depending on the substituents referred to as “X” in FIGS. 6 and 7 .
  • the molar ratio of cataylyst:alkynyl group:azido group is from 0:1:1 to 2:1:100, and preferably 1:1:0.5.
  • the reaction is carried out in the aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • FIG. 8 shows a scheme for the immobilization of a polysaccharide on a solid surface by 1,3-dipolar cycloaddition reaction.
  • the polysaccharide is labeled with an azido group at the terminal sugar ring, while the solid surface is modified by a heterobifunctional linker which produces a substituted alkynyl group at the end.
  • the polysaccharide is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • the positions of the azido and the alkynyl functional groups are interchangeable as similarly shown in FIGS. 6 and 7 .
  • the 1,3-dipolar cycloadditon reaction is controlled either thermodynamically at high temperature, or catalytically at room temperature with cucurbituril (21). In the absence of the catalyst the reaction is carried about within the temperature range 50° C. to 150° C., and more usually at between 70° C. to 100° C. The reaction takes from 5 hours to 7 days depending on the substituents referred to as “X” in FIGS. 6-9 .
  • the molar ratio of catalyst:alkynyl group:azido group is from 0:1:1 to 2:1:100, and preferably 1:1:0.5.
  • the reaction is carried out in the aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • FIG. 9 shows a scheme for the immobilization of a protein on a solid surface by 1,3-dipolar cycloaddition reaction.
  • the protein is labeled with an azido group, while the solid surface is modified by a heterobifunctional linker which produces a substituted alkynyl group at the end.
  • the protein is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • the positions of the azido and the alkynyl functional groups are interchangeable as similarly shown in FIGS. 6 and 7 .
  • the 1,3-dipolar cycloadditon reaction is controlled either thermodynamically at high temperature, or catalytically at room temperature with cucurbituril (21). In the: absence of the catalyst the reaction is carried about within the temperature range 50° C. to 150° C., and more usually at between 70° C. to 100° C. The reaction takes from 5 hours to 7 days depending on the substituents referred to as “X” in FIGS. 6-9 .
  • the molar ratio of catalyst:alkynyl group:azido group is from 0:1:1 to 2:1:100, and preferably 1:1:0.5.
  • the reaction is carried out in the aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • FIG. 10 shows a scheme for the immobilization of an oligonucleotide on a solid surface by 1,3-dipolar cycloaddition reaction.
  • the oligonucleotide is labeled with an azido group at the 5′ end, while the solid surface is modified by a heterobifunctional linker which produces a substituted alkynyl group as the terminal functional group.
  • the oligonucleotide is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • the positions of the azido and the alkynyl functional groups are interchangeable as similarly shown in FIGS. 6 and 7 .
  • the 1,3-dipolar cycloadditon reaction is controlled either thermodynamically at high temperature, or catalytically at room temperature with cucurbituril (21). In the absence of the catalyst the reaction is carried about within the temperature range 50° C. to 150° C., and more usually at between 70° C. to 100° C.
  • the molar ratio of catalyst:alkynyl group:azido group is from 0:1:1 to 2:1:100, and preferably 1:1:0.5.
  • the reaction is carried out in the aqueous phase or aqueous/water-soluble organic mixture such as water/dimethylformamide or water/methyl sulfoxide as the solvent system.
  • FIG. 11 shows a scheme for the immobilization of a DNA on a glass surface by 1,3-dipolar cycloaddition reaction in the presence of a Cu(I) catalyst.
  • the DNA is labeled with an azido group at the 5′ end, while the glass surface is modified by an alkynyl group.
  • the DNA is covalently attached to the surface via a stable 1,2,3-triazole linkage.
  • the positions of the azido and the alkynyl functional groups are interchangeable.
  • the amino-C6-M13 ( ⁇ 40) forward primer (18 mer) and the internal mass standard oligonucleotides were commercially available and purified by HPLC.
  • the 1H and 13 C NMR spectra were recorded on 400 MHz and 300 MHz NMR spectroscopic instruments, respectively.
  • the high-resolution mass spectra (HRMS) were obtained under fast atom bombardment (FAB) conditions.
  • UV-Vis spectra of the DNA samples were recorded in acetonitrile/water (1:1 volume ratio) at room temperature using quartz cells with path lengths of 1.0 cm.
  • succinimidyl 5-azidovalerate was synthesized according to the published procedure (18). 500 mg (2.61 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to a suspension of 358 mg (2.50 mmol) of 5-azidovaleric acid and 300 mg (2.61 mmol) of N-hydroxysuccinimide in CH 2 Cl 2 (20 mL) at room temperature and stirred for 7 h, followed by the addition of H 2 O.
  • EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • DNA immobilization on a glass surface using the 1,3-dipolar cycloaddition coupling chemistry The amino-modified glass (Sigma) surface was cleaned by immersion into a basic solution (dimethylformamide (DMF)/N,N-diisopropyl-ethylamine (DIPEA) 90/10 v/v) for 1 h, sonicated for 5 min, washed with DMF and ethanol, and then dried under air.
  • the precleaned glass surface was functionalized by immersing it into the terminal alkyne crosslinker solution (20 mM of succinimidyl N-propargyl glutariamidate in DMF/pyridine (90/10 v/v)) for 5 h at room temperature.
  • the glass slide was incubated in a humid chamber at room temperature for 12 h, then washed with dH 2 O, and SPSC buffer (0.25 M sodium phosphate, 2.5 M NaCl, pH 6.5) extensively for 1 h to remove nonspecifically bound DNAs (28), and finally rinsed with dH 2 O and ethanol.
  • Atomic force microscopy (AFM) and water contact angle measurement were used for the characterization of the change on the surface after each step in the immobilization process.
  • Mass spectrum of DNA Mass measurement of oligonucleotides was performed using a MALDI-TOF mass spectrometer. 30 pmol of the DNA product was mixed with 10 pmol of the internal mass standard and the mixture was suspended in 2 ⁇ L of 3-hydroxypicolinic acid matrix solution. 0.5 ⁇ L of this mixture was spotted on a stainless steel sample plate, air-dried and analyzed.
  • the measurement was taken using a positive ion mode with 25 kV accelerating voltage, 94% grid voltage and a 350 ns delay time.
  • a PCR DNA product amplified from a pBluescript II SK(+) phagemid vector was used as a sequencing template as it has a binding site for M13-40 universal primer.
  • Amplification was carried out using the M13-40 universal forward and reverse primers in a 20 ⁇ L reaction, which contained 1 ⁇ ACCUTAQ LA Reaction Buffer, 25 pmol of each dNTP, 40 pmol of each primer, 0.5 unit of Jumpstart Red ACCUTAQ LA DNA Polymerase and 100 ng of the phagemid template.
  • the reaction was performed in a DNA thermal cycler using an initial activation step of 96° C. for 1 minute. This was followed by 30 cycles of 94° C. for 30 seconds, 50° C.
  • a primer extension reaction was performed using the FAM-labeled primer “4” and “5” and the above PCR product.
  • a 30 ⁇ L reaction mixture was made, consisting of 2.22 nmol of each dNTP, 37 pmol of Biotin-11-ddATP, 20 pmol of primer, 9 units of Thermo Sequenase DNA polymerase, 1 ⁇ Thermo Sequenase Reaction Buffer and 20 ⁇ L of PCR product.
  • the reaction consisted of 30 cycles of 94° C. for 20 seconds, 50° C. for 20 seconds and 60° C. for 90 seconds.

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WO2004055160A2 (fr) 2004-07-01

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